Synergy of antioxidant and M2 polarization in polyphenol‐modified konjac glucomannan dressing for remodeling wound healing microenvironment

Abstract Effective skin wound healing and tissue regeneration remain a challenge. Excessive/chronic inflammation inhibits wound healing, leading to scar formation. Herein, we report a wound dressing composed of KGM‐GA based on the natural substances konjac glucomannan (KGM) and gallic acid (GA) that accelerates wound healing without any additional drugs. An in vitro study showed that KGM‐GA could not only stimulate macrophage polarization to the anti‐inflammatory M2 phenotype but also decrease reactive oxygen species (ROS) levels, indicating excellent anti‐inflammatory properties. Moreover, in vivo studies of skin wounds demonstrated that the KGM‐GA dressing significantly improved wound healing by accelerating wound closure, collagen deposition, and angiogenesis. In addition, it was observed that KGM‐GA regulated M2 polarization, reducing the production of intracellular ROS in the wound microenvironment, which was consistent with the in vitro experiments. Therefore, this study designed a multifunctional biomaterial with biological activity, providing a novel dressing for wound healing.


| INTRODUCTION
Skin tissue is the natural barrier that protects the body from environmental damage and microbial infestation. 1 When the skin is damaged, pathogens are more likely to invade the body and cause inflammation and infection, affecting the process of wound healing. 2 Currently, several treatments, including growth factors [3][4][5] and stem cells, 6,7 can drive wound healing, but these approaches are limited by their high cost and side effects. 8 However, there have been few studies on regulating the wound microenvironment to utilize the inherent regenerative capacity of the host. Most treatment methods mainly focus on the process of structural repair, and nonfibrotic healing of damaged tissues is still difficult to achieve. 9 Effective restoration of wound tissue integrity and function remains a global health issue.
Macrophages, which play an important role in tissue repair, are highly plastic cells and can be polarized into classic M1 type (inflammatory phenotype in the early stage) and M2 type (anti-inflammatory phenotype in the mid-stage), and these cells can shift between phenotypes under certain conditions. 10,11 M2 macrophages have been shown to secrete a range of anti-inflammatory factors, such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and secrete healing factors that reduce the inflammatory response, promote angiogenesis, and create a regenerative microenvironment. 12 Appropriate regulation of the M1-to-M2 shift is critical in promoting tissue repair and coordinating skin healing. Studies have confirmed that cytokines, such as interleukin-4 (IL-4) and interleukin-13 (IL-13), 13,14 can be used to polarize M2 macrophages. However, these recombinant proteins are unstable in the body and difficult to effectively deliver, and excessive use may cause serious side effects, thus limiting their application. Konjac glucomannan (KGM) is a natural polysaccharide that consists of D-glucose and D-mannose linked by a β-1,4 glycosidic chain. 15 The abundant carbohydrate receptors expressed on the surface of macrophages interact with these sugar units to activate murine monocytes/macrophages, such as the mannose receptor (MR), which responds to mannan. 16 In addition, KGM has also been reported to be nontoxic, biodegradable, and biocompatible. 17 Moreover, KGM exhibits outstanding liquid adsorption capacity owing to its structure, which contains plentiful hydroxyl and carboxyl groups that can attract water molecules through hydrogen bonding and van der Waals forces. 18 These excellent properties of KGM are conducive to its application in wound healing.
In addition to regulating macrophage phenotype to resist inflammation, controlling the level of reactive oxygen species (ROS) in impaired wounds has become another research direction. Many studies have shown that ROS play an important role in the wound microenvironment. [19][20][21] After skin injury, the wound surface produces abundant ROS, which is one of the defense mechanisms against bacterial infections. 22 However, high levels of ROS, such as hydrogen peroxide (H 2 O 2 ), can cause oxidative stress in the impaired wound and trigger a series of harmful effects, such as cell aging, 23 fibrotic scarring, 19 and inflammation. 24 In addition, ROS can significantly limit angiogenesis, leading to endothelial dysfunction. 20 ROS can also inhibit the function of endogenous stem cells and macrophages, hinder the regeneration of wound tissue, and ultimately impede the process of wound healing. 22,25 Therefore, studying biomaterials that have the ability to eliminate ROS and control oxidative damage in the microenvironment of impaired wounds is a potential treatment strategy to promote wound healing. Gallic acid (GA) belongs to a class of natural polyphenol compounds that are widely found in the plant kingdom. 26 GA has potential anti-inflammatory and antioxidant effects and can directly upregulate the expression of antioxidant genes. Previously, GA was used in traditional medicines for the treatment of various chronic skin diseases, such as psoriasis and vitiligo. 27,28 Recent studies have shown that GA promotes wound healing and accelerates the migration of keratinocytes and fibroblasts. 29 In addition, due to its excellent antioxidant properties and lack of toxicity, GA can be used to modify functional materials to improve their physical, chemical, and biological properties. 30 This study prepared a polymer dressing designed to improve the wound microenvironment, which could regulate the conversion of macrophages to the M2 phenotype while controlling the level of ROS in the impaired wound (Scheme 1). In this study, KGM was conjugated with GA by esterification to prepare a material for skin repair. Our research results showed that in the absence of any exogenous cytokines or drugs, KGM-GA could significantly upregulate M2 macrophage polarization and eliminate excess ROS, which may provide insight into the development of efficient dressings for improving the wound healing process. (h) Evaluation of in vivo toxicity of KGM-GA to major organs (heart, liver, spleen, lung, and kidney) at 14 days after treatment compared with normal mice (control group). The data were conducted by two-way ANOVA with Sidak's post hoc test. The data are represented by means ± SD (n = 3, ns p > 0.05).

| KGM-GA synthesis and structural characterization
The reaction of KGM and GA occurred between the hydroxyl group of KGM and the carboxylic group of GA to form the ester linkage using the EDC/DMAP-mediated coupling reaction. The procedure to synthesize KGM-GA is illustrated in Scheme 1. The structure of KGM-GA was first confirmed by Fourier transform infrared (FTIR). Figure 1a shows the FTIR spectra of KGM and GA-KGM. In the FTIR spectrum of KGM, the absorption band of the carbonyl of the acetyl groups was observed at 1727 cm À1 . The intense peak at 1632 cm À1 was attributed to the in-plane deformation of the water molecule. In contrast to the spectrum of KGM, the band at approximately 1727 cm À1 was also ascribed to the carbonyl, which changed from a small shoulder in pure

| Biocompatibility of KGM-GA
The cytotoxicity of KGM, GA, and KGM-GA toward Raw 264.7 and L929 cells was evaluated using CCK-8 and live/dead cell staining assays. As shown in Figure 1d and Figure S1, within a certain concentration range, none of the groups showed obvious cytotoxicity, and the viability of Raw 264.7 cells exceeded 80% and was 95% for L929 cells. Moreover, cell viability was examined using live and dead cell staining in the KGM-GA group ( Figure S2). The majority of cells were green, and at the test concentrations, there was no noticeable cytotoxicity. Next, the effect of KGM-GA on the hemolysis of red blood cells was studied. As shown in Figure S3, 1 mg/ml KGM-GA affected the integrity of erythrocytes in the same way as the control PBS buffer (negative group), indicating good biocompatibility.
The in vivo biological safety of a material is a key factor in its application. Next, we evaluated the effects of KGM-GA on mouse blood chemistry and major organ histopathology to determine in vivo biocompatibility. As shown in Figure 1e-g, complete blood panel analysis showed no obvious differences in hematology at 14 days after treatment with KGM-GA in wounded mice compared to that of the control (p > 0.05). Moreover, no necrosis, congestion, or hemorrhage was observed in the heart, liver, spleen, lung, or kidney ( Figure 1h).
These results demonstrated the excellent biocompatibility of KGM-GA in vivo.

| Regulation of macrophage polarization in vitro
Macrophages are essential regulators of wound healing and are known to have many functions, including participating in inflammation and promoting tissue repair and regeneration. 31 According to their activation states and functions, they can be divided into M1 and M2 phenotypes, which play different roles in wound healing. Despite M1 cells playing a critical role in defending against external pathogens in the early stage, 32 chronic M1 activation and continuous secretion of proinflammatory cytokines delay wound healing. 33 In contrast, M2 macrophages can secrete anti-inflammatory cytokines and extracellular matrix components, which are necessary for the late stage of tissue repair, 34 and studies have shown that the infiltration of M2 macrophages reduces scar formation. 35 First, to evaluate the effects of different concentrations of KGM-GA on the polarization of macrophages, we performed flow cytometric analysis of the M1 marker CD86 and M2 marker CD206 in Raw 264.7 cells. As shown in Figure S4, the expression of CD206 increased in response to KGM-GA in a concentration-dependent manner. However, at a concentration of 100 μg/ml, the expression of CD86 also increased significantly. Then, we calculated the ratio of CD206 to CD86 and found that the maximum occurred at a concentration of 50 μg/ml KGM-GA, which was used in subsequent experiments ( Figure S5).
Morphological changes in macrophages are related to their functional polarization. Previous studies used different signals to stimulate morphological changes in macrophages from a round shape to an elongated shape, which tended to be M2 macrophages. 36 Figure 2b, and the formula for calculating elongation was described in previous research. 38 As shown in Figure 2c, the control group exhibited an elongation of 1.15 ± 0.18, while the KGM and KGM-GA groups exhibited elongations of 2.15 ± 0.7 and 2.77 ± 0.82, respectively, which were significantly different from those in the control group. As demonstrated by many studies, enhanced macrophage elongation is believed to be closely related to the M2 phenotype.
To further evaluate the effects of KGM-GA on M2 polarization, we examined the expression of M1 and M2 macrophage markers. Immunofluorescence staining revealed that after 48 h of incubation with KGM-GA, the expression of the M2 surface marker CD206 was significantly increased, and the intensity was greater than that induced by KGM ( Figure 3a,b). In addition, these results suggested that KGM-GA was a potent driver of M2 polarization without significantly stimulating the transformation of cells to the M1 phenotype. Quantitative analysis was followed by flow cytometry. As shown in Figure 3c,d, after KGM-GA treatment, the percentage of cells expressing CD206 among total cells was 20.98% ± 4.1%, which was nearly 5.4 times higher than those in the control group, more than 3.8 times higher than those in the GA group, and approximately 2.2 times than those in the KGM group. In addition, we found that all groups expressed low levels of CD86, and there was no significant difference among the groups. Moreover, some cells coexpressed CD86 and CD206, indicating a transitional state between the M1 and M2 phenotypes in these macrophages. We examined the expression of M1 and M2 macrophage markers.

| ROS scavenging activities of KGM-GA in vitro
The potential antioxidant activity of KGM-GA was evaluated in this study ( Figure 4a). Free radicals and H 2 O 2 were selected as representative ROS to examine the ROS scavenging activities of KGM-GA by DPPH and Amplex Red assays, respectively. As shown in Figure  Moreover, the intracellular ROS levels were also quantitatively analyzed by flow cytometry (Figure 4h,i). The relative percentage ROS in L929 cells was reduced from nearly 35% to less than 20% after treatment with KGM-GA. KGM slightly eliminated intracellular ROS levels in L929 cells, which may be related to KGM promoting the proliferation of fibroblasts by stimulating metabolism in cells, as reported. 17 KGM was modified with GA, and KGM-GA exerts an excellent effect against oxidative stress, which is highly suitable for wound healing and regeneration.

| Cell migration and proangiogenic responses in vitro
Fibroblasts play a vital role in tissue repair. To evaluate the effect of KGM-GA on fibroblasts, the migration of L929 cells was examined. As Tests were conducted by oneway ANOVA with Tukey post hoc analysis. The data are presented as the mean ± SD (n = 3). ***p < 0.001, **p < 0.01, and *p < 0.05 compared to control group shown in Figure 5a, incubation with a certain concentration of H 2 O 2 affected the migration of cells compared with those in the normal group. However, cells exposed to KGM, GA, and KGM-GA showed a significant increase in migration compared with those in the control groups. After 12 h, cells exposed to KGM-GA showed the highest migration (43.6% ± 2.4%), followed by those exposed to PBS (5.1% ± 7.3%), KGM (31.4% ± 2.0%), and GA (31.6% ± 7.1%) (Figure 5c). In addition, after being incubated with different materials for 48 h, the level of VEGF in the cell supernatant was measured. As shown in Figure 5d, KGM-GA obviously improved the secretion of VEGF, suggesting that KGM-GA promotes angiogenesis.
Since angiogenesis is a key process in skin tissue repair, we next evaluated the ability of KGM-GA to drive angiogenesis in HUVECs. In a hydrogen peroxide environment, the vessel-forming capability of HUVECs was examined by the tube formation assay. As shown in Although sporadic tube formation was observed, the length was short, and the tubes did not form a dense network structure, indicating that the formation of blood vessels was blocked in the ROS environment.   Figure S6). Furthermore, the average length of the wound edge was calculated and is shown in Figure 6e. The length in KGM-GA group was significantly smaller than that in the control group (Figure 6d). In addition, the results of picrosirius red staining showed that collagen deposition in the KGM-GA group was higher (red staining) and collagen fibers were arranged more regularly than in the control group (Figure 6f). CD31 is a vascular endothelial growth factor that can promote angiogenesis. An anti-CD31 antibody was used to stain newly formed blood vessels in the wound. On Day 7, the count of capillary densities showed that a significantly higher density of capillaries was produced in KGM-GA ( Figure S7).
Moreover, after 14 days of treatment (Figure 6g

| DISCUSSION
The repair and regeneration of skin injuries caused by traumatic injury, surgery, or disease remain major clinical challenges and contribute to increased health care costs. 39 Wound healing is a dynamic, intertwined, and complex process that includes multiple stages, and the prolongation of the inflammatory period adversely affects subsequent tissue regeneration. 40 On the one hand, excessive inflammation at the damaged tissue site prolongs the inflammation period and delays the wound healing process, which may lead to the development of pathological fibrosis or scarring, thereby destroying normal tissue structure and function. 41 On the other hand, delayed wound healing will greatly increase the risk of infection, which further aggravates the healing process and leads to a vicious cycle. 40 Therefore, effective therapies for healing wounds targeting the inflammatory microenvironment of the injured site should be considered. It has been widely acknowledged that macrophages are important regulators of the wound healing process and are involved in advancing inflammation and promoting tissue repair and regeneration. 32 Many studies have shown that the conversion of proinflammatory macrophages (M1 type) to an anti-inflammatory phenotype (M2 type) is critical for normal wound repair and fibrosis reduction. 42 In addition, inflammation is intimately associated with oxidative stress. 43 An excessive inflammatory response at the wound site results in the production of a large amount of ROS, 44,45 which can aggravate local tissue damage, reduce the migration and proliferation of fibroblasts, keratinocytes, and endothelial cells, and delay wound healing. 46 Recent studies have focused on unilateral regulation of macrophage polarization or the control of oxidative stress. A variety of biologically active substances have been used to modulate the phenotype of macrophages, including related cytokines, 47,48 receptors, 49 small molecules, 50 and mesenchymal stem cells. 10,40 However, their application is hindered because of uncontrollable biological activity. The use of biological materials to regulate cell fate and activity may lead to the development of new therapeutic strategies.
Here, the natural polysaccharide KGM has attracted our attention due to its ability to regulate macrophage polarization and exposed chemical structures that are easily modified. 16,51 Recently, hydrolysates of Tests were conducted by one-way ANOVA with Tukey post hoc analysis. The data are presented as the mean ± SD (n = 6). ***p < 0.001 compared to control group; ##p < 0.01 compared to GA group, ###p < 0.001 compared to KGM group to and proliferation within the wound site are prerequisites for wound granulation during the proliferation stage, and angiogenesis is an indispensable step in the remodeling phase during wound healing. 55 Excessive ROS accumulation inhibits fibroblast migration and angiogenesis, which was also confirmed in our experiments. Further statistical analysis revealed that KGM-GA downregulated inflammation and improved oxidative stress to create an environment for the migration of L929 cells and tube formation by HUVECs.
Prior studies have shown that inherent contraction of skin wounds can only induce closure of the epidermis but cannot promote regeneration of the intact epidermal layer and mature dermis. 19 This study confirmed that the application of KGM-GA not only promoted wound healing but also improved skin tissue regeneration. Notably, KGM-GA  56 Collectively, these results demonstrated that KGM-GA could create a beneficial microenvironment for promoting tissue recovery and regeneration.
In most situations, wound repair will face unexpected challenges due to exposure to pathological conditions, such as aging, obesity, and diabetes. In diabetic wounds, macrophages exhibit a reduced capability to induce the phenotypic switch from M1 to M2 due to hyperglycemia and the presence of excessive glycosylation residues, resulting in accumulation and enrichment of M1 macrophages, causing excessive inflammation. 57,58 Therefore, wound healing in diabetes requires modulation of the polarization state of macrophages to reduce local inflammation. Currently, some studies have used polysaccharides to regulate macrophage phenotype to treat diabetic wound healing and have achieved certain therapeutic effects. [59][60][61] In addition, excessive ROS accumulated in diabetic wounds hinders the regeneration of wound tissue. 19,20 Furthermore, in recent decades, inflammatory signals have not only been thought to influence wound healing but are also driving factors for diseases such as atherosclerosis 62,63 and cancer. 64 The polymer we synthesized can regulate macrophages and combat oxidative stress without the aid of cytokine or gene delivery. The insights gained from this study may be of assistance in    RPMI-1640 medium containing 10% fetal bovine serum (FBS) was used for L929 cell maintenance, and Dulbecco's Modified Eagle Medium containing 10% FBS was used for Raw264.7 cell maintenance. The cells were seeded in 96-well plates (5000 cells/well) and incubated for 24 h at 37 C in a 5% CO 2 atmosphere. Then, the cells were incubated with a series of concentrations of KGM, GA, and KGM-GA for 24 h. Cell viability was evaluated by CCK-8 cell proliferation and cytotoxicity assay kits (Solarbio, CA1210), and the absorbance at 450 nm was measured using a microplate reader (Thermo).

| Cytotoxicity and blood compatibility assay
Moreover, a live/dead cell staining kit (Solarbio, CA1630) was used to stain the cells. Finally, the images of the cells were observed by a fluorescence microscope from the Nikon Corporation (Tokyo, Japan).
The effect of KGM-GA on erythrocyte hemolysis was examined according to a previously reported method. 65 In brief, blood was collected from the orbital sinus and centrifuged at 3000 rpm for 15 min. In addition, VEGF expression in L929 cells was measured by ELISA. In brief, the cells were seeded in 24-well plates (1 Â 10 5 cells/ well) and treated with KGM, GA, or KGM-GA for 48 h. Finally, the supernatants were collected to measure VEGF levels.

| Wound healing in vivo
The therapeutic effect of KGM-GA on wound healing was evaluated using a wound model. Female BALB/C mice (18-21 g, 6-7 weeks) were anesthetized by 4% chloral hydrate, and then the hair on the dorsal skin was shaved. Full-thickness circular wounds with diameters of 6 mm were created by surgical procedures on the backs of the mice. The mice were randomly divided into four groups (n = 6) and treated with 50 μl of a saline solution dispersion of KGM, GA, or KGM-GA (20 mg/ml) at the wound site. The other mice were treated with saline solution as controls. Images of the wounds were captured at Days 0, 3, 7, and 14 post-wounding using a digital camera.

| Histological analysis
After 7 and 14 days, the wound tissues were collected from the mice, fixed with 4% formaldehyde, and then embedded in paraffin. Then, the paraffin-embedded tissues were sectioned into 6 μm thick slices for hematoxylin and eosin (H&E) and picrosirius red staining.

| Immunofluorescence analysis
The skin sections were deparaffinized and rehydrated and then

| ROS measurement in wound sites
The wound specimens at Day 7 post-wounding were washed with PBS three times. After that, the skin sections were incubated with 2.5 μM dihydroethidium (DHE) at 37 C for 30 min and imaged with a fluorescence microscope.

| In vivo biocompatibility of KGM-GA
At 14 days of treatment, blood samples were collected for complete blood panel analysis. In addition, major organs, including the heart, liver, spleen, lung, and kidney, were removed, and paraffin sections were prepared. HE staining was performed to assess tissue structure.
All data are compared with normal mice.

| Statistical analysis
The data are presented as the mean ± standard deviation. Where appropriate, a one-way or two-way analysis of variance (ANOVA) was performed to assess statistical significance. Statistical evaluations were performed using GraphPad Prism 8.0, and p values <0.05 were considered statistically significant.

| CONCLUSION
In this study, we present a simple and efficient technique to synthe-